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Integration of large heterologous DNA fragments into the genome of Thermococcus kodakarensis

Abstract

In this study, a transformation system enabling large-scale gene recombination was developed for the hyperthermophilic archaeon Thermococcus kodakarensis. Using the uracil auxotroph T. kodakarensis KU216 (∆pyrF) as a parent strain, we constructed multiple host strains harboring two 1-kbp DNA regions from the genomes of either the hyperthermophilic archaeon Pyrococcus furiosus or Methanocaldococcus jannaschii. The two regions were selected so that the regions between them on the respective genomes would include pyrF genes, which can potentially be used for selection. Transformation using these host strains and genomic DNA from P. furiosus or M. jannaschii were carried out. Transformants with exogenous pyrF were obtained only using host strains with regions from P. furiosus, and only when the distances between the two regions were relatively short (2–5 kbp) on the P. furiosus genome. To insert longer DNA fragments, we examined the possibilities of using P. furiosus cells to provide intact genomic DNA. A cell pellet of P. furiosus was overlaid with that of T. kodakarensis so that cells were in direct contact. As a result, we were able to isolate T. kodakarensis strains harboring DNA fragments from P. furiosus with lengths of up to 75 kbp in a single transformation step.

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Abbreviations

PyrF:

Orotidine-5′-monophosphate decarboxylase

ASW:

Artificial seawater

YT:

Yeast extract and tryptone

AA:

20 Amino acids

5-FOA:

5-Fluoroorotic acid

CFE:

Cell-free extract

GAP:

Glyceraldehyde 3-phosphate

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Acknowledgements

The authors are grateful to Ms. Rikako Fujimoto for constructing the expression plasmid pPcsgChiA1. This work was partially supported by JSPS KAKENHI Grant Number JP19H05679 (Post-Koch Ecology), JP19H05684 to H.A. and JP19H05689 to M.O., and initiated with the support of the CREST program of the Japan Science and Technology Agency (Grant No.: 10104047) to H.A. This work was also partially supported by JSPS KAKENHI to T.S. (Grant No.: 18K05411).

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Correspondence to Haruyuki Atomi.

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Sato, T., Takada, D., Itoh, T. et al. Integration of large heterologous DNA fragments into the genome of Thermococcus kodakarensis. Extremophiles 24, 339–353 (2020). https://doi.org/10.1007/s00792-020-01159-z

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Keywords

  • Archaea
  • Hyperthermophile
  • Gene recombination
  • Large-scale gene cloning